A number of 5'-terminal 'cap' structure analogs have been synthesized, including m7GppA, GppA, GpppG and GpppppG, while their structural characterizations have been achieved through 31P-NMR spectral analysis. A new method for the assay of poly (A) polymerase and ATP analog activities has been achieved using the luciferin-luciferase (Firefly) enzyme coupling system. 8N3-ATP, 8Br-ATP and 2'-deoxy ATP have been shown to be active in both the poly (A) polymerase and the luciferin-luciferase enzymatic systems. We have further shown that 8-azido ATP can be added to the 3'-end of poly (U) mRNA and natural mRNAs, such as 9s rabbit globin. A number of mononucleotides such as m7GMP, m7GDP are known to inhibit protein synthesis, presumable through competing with mRNA for its binding site(s). Through various experiments, we have shown that translation of poly (U) and 9s rabbit globin mRNA is inhibited by S4UMP and S4UDP, in both wheat germ and rabbit eticulocyte protein synthesizing systems. Consequently, we believe that these photoaffinity labeled compounds will be useful tools in elucidating the role of the 5'-end capped and uncapped mRNAs in protein biosynthesis. We intend to utilize these probes in detecting mRNA binding proteins of ribosomal or non-ribosomal origin. We propose to synthesize 'cap' analogs, m7GvpppN*, where N* indicates either a photoaffinity label or a spin label and v represents protected 2' and 3' hydroxyls. Upon successful preparation of these compounds, we will initially study their inhibition of protein synthesis in a wheat germ cell-free system to see if they will compete with mRNA for the same site. They will then be incorporated into synthetic mRNA with the aid of T4RNA ligase. Conformation of all synthetic 'cap' analogs will be analyzed through 3IP-NMR analysis, molecular orbital calculations and Laser Raman analysis.